The collagen receptor DDR1 co-localizes with the non-muscle myosin IIA in mice inner ear and contributes to the cytoarchitecture and stability of motile cells.

Discoidin domain receptor 1 (DDR1) is a tyrosine kinase receptor activated by native collagen. DDRs regulate cell adhesion, migration and various other cell functions. Deletion of the DDR1 gene in mice is associated with a severe decrease in auditory function and substantial structural alterations in a heterogeneous group of cells, including cells containing actin/myosin contractile elements, e.g., outer hair cells (OHCs) (Meyer zum Gottesberge et al. Lab Invest, 88: 27-37, 2008). The non-muscle myosin heavy chain isoform IIA (NM-IIA), encoded by MYH9, is implicated in the regulation of cell spreading, cellular reshaping and movement and cell migration and adhesion. In this study, we identify DDR1 and NM-IIA co-localization in the type III fibrocytes (tension fibrocytes) of the spiral ligament, the OHCs and the stereocilia of both OHCs and inner hair cells. We show for the first time that DDR1 malfunction causes OHC deformation and the separation of the lateral wall, the location of the cellular motor responsible for the electromotile property, explicitly in those regions showing DDR1 and NM-IIA co-localization. On the basis of our results, we propose that DDR1 acts in concert with proteins of the actin/myosin complex to maintain mechanical forces in the inner ear and to stabilize OHC cellular shape for proper auditory signal transduction.

Ultrastructural and physiological defects in the cochlea of the Mpv17 mouse strain. A comparison between young and old adult animals.

Ultrastructural investigations were performed in young (approximately 2 months) and old (7 months) Mpv17-negative and wild-type mice. The onset, the severity and the pattern of the degeneration significantly differed between both mice strains. In the wild-type mouse strain the degenerative changes of the cochlear structures were similar to the aging pattern described for other species. In contrast, the Mpv17 mutants showed degenerative changes of the cochlear structures already at the age of 2 months. The degenerative changes were patchy arranged throughout the entire length of the cochlea and involved the organ of Corti as well as the stria vascularis epithelia with alterations of the basement membrane of the capillaries. The severe sensorineural hearing loss and degenerative changes of the cochlear structures indicate that cochlear structures, especially the outer hair cells and the intermediate cells of the stria vascularis, are vulnerable to the missing Mpv17 gene product.

Expression of the recessive glomerulosclerosis gene Mpv17 regulates MMP-2 expression in fibroblasts, the kidney, and the inner ear of mice.

The recessive mouse mutant Mpv17 is characterized by the development of early-onset glomerulosclerosis, concomitant hypertension, and structural alterations of the inner ear. The primary cause of the disease is the loss of function of the Mpv17 protein, a peroxisomal gene product involved in reactive oxygen metabolism. In our search of a common mediator exerting effects on several aspects of the phenotype, we discovered that the absence of the Mpv17 gene product causes a strong increase in matrix metalloproteinase 2 (MMP-2) expression. This was seen in the kidney and cochlea of Mpv17-negative mice as well as in tissue culture cells derived from these animals. When these cells were transfected with the human Mpv17 homolog, an inverse causal relationship between Mpv17 and MMP-2 expression was established. These results indicate that the Mpv17 protein plays a crucial role in the regulation of MMP-2 and suggest that enhanced MMP-2 expression might mediate the mechanisms leading to glomerulosclerosis, inner ear disease, and hypertension in this model.

Transcripts encoding three types of guanylyl-cyclase-coupled trans-membrane receptors in inner ear tissues of guinea pigs.

The distribution of membrane-bound guanylyl cyclase (GC) transcription in inner ear tissues of the guinea pig was addressed by a reverse transcription-PCR approach using consensus primers flanking a region of about 630 bp in the intracellular domains in the target sequences. Restriction mapping of such amplificates obtained from cochlear and vestibular specimens permitted us to demonstrate GC-A, GC-B, and GC-C expression by differentiating overall PCR signals. This assay indicated that GC-A was expressed in the cochlea and vestibular organ. PCR products resulting from transcripts of the GC-B gene were obtained at considerably lower abundance than amplificates typical of the GC-A gene. The consensus primer approach with subsequent restriction mapping provided the opportunity to examine at the same time expression of GC-C in the inner ear and revealed the occurrence of GC-C transcripts in both inner ear compartments under investigation. The distribution pattern found by analysing the intracellular domains of membrane-bound guanylyl cyclases was confirmed by demonstrating transcription of the corresponding extracellular receptor domains. In addition, single-strand conformation polymorphism analysis of cDNA amplificates comprising the catalytic domain of guanylyl cyclases also indicated the presence of GC-C expression in the inner ear tissues examined. The GC-C transcripts detected in inner ear tissues appeared to correlate with functional receptor expression, since the production of cyclic GMP catalyzed by cochlear and vestibular specimens was stimulated by 1 microM of heat-stable enterotoxin to 18 and 80% above basal levels, respectively. Thus, GC-C may be involved in the fluid regulation by typical ligands (e.g., the peptide hormone guanylin or the toxins causing travellers' diarrhea), not only in the intestine but also in the organs responsible for hearing and gravitational orientation.

Melanin formation in the inner ear is catalyzed by a new tyrosine hydroxylase kinetically and structurally different from tyrosinase.

Detergent solubilized extracts of the cochleae of adult gerbils (Meriones unguiculatus) contain a tyrosine hydroxylase activity measurable by the radiometric method of Pomerantz. This activity is not related to Fenton-type reactions, since it is not inhibited by free radical scavengers and is heat and protease sensitive. It does not appear to be related to a peroxidase (EC 1.11.1.7) since it is neither dependent on H2O2, nor inhibited by catalase (EC 1.11.1.6). The involvement of a tyrosine hydroxylase (EC 1.14.16.2) related to catecholamine synthesis is also unlikely, since the activity is highly sensitive to 2-mercaptoethanol and is not increased by addition of tetrahydrobiopterin. The activity in crude inner ear extracts displayed an unusual maturation behaviour, with a slow activation upon aging at 4 degrees C. Fully active enzyme displayed Michaelis-Menten kinetics, with a Km for L-tyrosine of 47 microM. Cochlear tyrosine hydroxylase, but not melanoma tyrosinase (EC 1.14.18.1), was inhibited by o-phenanthroline, and was not dependent on L-DOPA as cofactor for full enzymatic activity. Crude extracts were also able to catalyze L-DOPA oxidation and melanin formation from either L-tyrosine or L-DOPA. The tyrosine hydroxylase, DOPA oxidase and melanin formation activities most probably resided in the same molecule, as suggested by inhibition studies. A tyrosine hydroxylase and melanin formation activity with identical properties was found in primary cultures of stria vascularis melanocytes. Immunochemical evidence confirmed the absence of either the tyrosinase encoded for by the albino locus, or the tyrosinase isoenzyme TRP1, encoded for by the brown locus. Conversely, an immunorreactive band of molecular weight 70 kDa was specifically recognized by a tyrosinase polyclonal antiserum in Western blot experiments. These results prove that melanogenesis in the cochlea, and likely in other extracutaneous locations such as the brain, is catalyzed by enzymatic systems different from, but related to tyrosinase.

Pre- and postnatal expression of glycoconjugates (3-fucosyl-N-acetyllactosamine and HNK-1 epitopes) in the mouse inner ear.

The distribution of two glycoconjugates 3-fucosyl-N-acetyllactosamine (CD15) and HNK-1 epitope (CD57) in the inner ear of the NMRI mouse was analysed from the eighth day of gestation to the 16th day after birth. CD15 epitope distribution is developmentally regulated. The up- and down-regulation of expression, the change in the number of cells which are positive, the ingrowth of CD15-positive cells and their distribution, intracellular and/or cell-surface-associated expression, all assume a characteristic appearance at each developmental stage. Distribution of CD57 documented the nerve outgrowth and formation of the innervation of the vestibular apparatus and cochlear duct. Correlation between CD15 and CD57 expression patterns revealed differences in the interaction of the ingrowing fibres and epithelial tissue between the vestibular organ and the cochlea and differences in the development of the cristae and maculae.

Glycerol administration modulates the pattern of the 3-fucosyl-N-acetyl-lactosamine (CD15) epitope in the adult guinea pig inner ear.

The effect of glycerol administration on the distribution pattern of the CD15 (3-fucosyl-N-acetyl-lactosamine) epitope has been studied immunohistochemically in hydropic and non-hydropic ears of adult guinea pigs from 80 min to 5 h after treatment with 2 g/kg glycerol. Glycerol administration characteristically modulated the immunoreactivity of the CD15 epitope in the endolymphatic sac and the tectorial membrane (TM) and revealed the CD15 epitope in the otoconia of the saccule, the stereocilia and the supporting cells of the ampullae. Our results suggest that glycerol interacts with glycoconjugates of the TM and otoconia, causing changes in their density and possibly the hydration state. As a consequence, the transduction process may be affected.

Loss of auditory function in transgenic Mpv17-deficient mice.

The transgenic mouse strain Mpv17 develops severe morphological degeneration of the inner ear and nephrotic syndrome at a young age (Meyer zum Gottesberge et al., 1996; Weiher et al., 1990). The audiograms (1-32 kHz) of Mpv17-negative mice were determined from auditory brain stem responses in young (2 months) and old (7 months) animals. Audiograms of age-matched wild-type mice with the same genetic background, but wild-type at the Mpv17 locus, were also determined. Furthermore, young Mpv17-negative mice that carried a human Mpv17 homologue gene were studied. NMRI mice served as a reference for normal hearing. Mpv17-negative mice suffer from severe sensorineural hearing loss as early as 2 months after birth. In the old Mpv17-negative mice no responses could be elicited at all. The 2 month old wild-type mice had normal audiograms, at 7 months only high threshold responses were seen. The poor audiograms of the Mpv17-negative mice are assumed to be the functional correlate of the morphological degeneration of the cochlea described earlier (Meyer zum Gottesberge et al., 1996). The finding that 2 out of 4 Mpv17-negative mice with the human Mpv17 gene had normal audiograms, shows that the gene inactivation can be functionally compensated by the human Mpv17 gene product.

Time-dependent alterations of 3-fucosyl-N-acetyl-lactosamine (CD15) expression in the endolymphatic sac of adult guinea pigs after glycerol administration.

The present study examined the effect of glycerol administration on 3-fucosyl-N-acetyl-lactosamine (CD15) epitope expression in the endolymphatic sac (ES) of the guinea pig's inner ear. Adult guinea pigs were injected intravenously with glycerol (2 g/kg body wt.). CD15 expression was studied at 80 min up to 5 h after treatment. In untreated animals single cells and cell groups in the ES expressing CD15 epitope intra- and intercellularly were identified by immunohistochemistry to be mainly in the epithelial layer of the rugosal and distal part of the sac. Glycerol administration modulated the expression of CD15 epitope. In the epithelial layer, expression decreased and was nearly depleted after 3 h. After 4 h of glycerol administration, CD15 expression reappeared and reached the comparable level of controls. The numbers of CD15-positive cells in the lumen of the ES increased steadily and arrived at their the highest levels in 2-h specimens. The localization of CD15-epitope expression and its modulation after glycerol administration within the ES implies that this molecule may play a role in re-establishing the sac's normal function. In addition, we speculate that CD15 may be associated with processes of an immune response in the inner ear.

Inner ear defect similar to Alport's syndrome in the glomerulosclerosis mouse model Mpv17.

The Mpv17 mouse strain is a recessive transgenic mouse mutant that develops glomerulosclerosis and nephrotic syndrome at a young age. The phenotype results from a loss of function of a gene coding for a hydrophobic peroxisomal protein of 176 amino acids of 20 kDa following its destruction by retroviral integration. To investigate a potential effect of the missing Mpv17 function on the inner ear light and electron microscopic investigations were performed on the inner ears of Mpv17 mice and controls. These revealed degeneration of the stria vascularis and spiral ligament, loss of cochlear neurons and degeneration of the organ of Corti. The alterations observed here were similar to those described for Alport's syndrome, an inherited disorder characterized by progressive nephritis and neurosensory deafness. These findings indicate that although the molecular cause is different, the Mpv17 mouse model may share pathological mechanisms involved in patients with Alport's syndrome. At present the Mpv17 mouse appears to be a suitable animal model for this disease and may help to further elucidate the relationship between the kidney and the inner ear.

[Dimorphism of cerumen, facts and theory]. / Der Dimorphismus des Cerumens, Fakten und Theorie.

¿Dimorphism¿ means the existence of two phenotypic different types of the human cerumen and morphology of the cerumen glands. In this paper, the morphological, biochemical, and functional differences are reviewed. The wet cerumen is better adapted to a hot and humid climate where infections of the outer ear are very frequent and sometimes severe. In a cold and dry climate, the dry cerumen is advantageous. In the moderate climates, both types are equivalent (balanced dimorphism).

Expression of the carbohydrate epitope 3-fucosyl-N-acetyl-lactosamine (CD 15) in the adult guinea pig inner ear.

The expression of the CD 15 (3-fucosyl-N-acetyl-lactosamine) epitope was immunohistochemically studied on paraffin sections of adult guinea pig inner ears. Two regions of the inner ear expressed the epitope for CD 15: the tectorial membrane of the cochlea and the endolymphatic sac. The upper part of the main body of the tectorial membrane was deeply stained. In the rugosal and distal part of the endolymphatic sac several unevenly distributed cells showed strong intra- and extracellular localization of the CD15 epitope. The CD15 epitope is associated with a transduction structure (tectorial membrane) and with a "volume regulating" compartment (endolymphatic sac) and may be involved in the maintenance of the structural integrity of both.

[Cerumen from the anthropologic viewpoint]. / Das Cerumen in anthropologischer Sicht.

The dimorphism of cerumen was first discovered by Kishi in 1907. There are two types of cerumen which differ in colour and consistency: the flocky and gray dry cerumen and the sticky yellow to brown wet cerumen. The comprehensive review of the frequency of the different cerumen types and their geographical distribution result in evidence for the dry type as a distinguishing feature of the mongoloid peoples (including the American Indians). The frequency of the wet cerumen prevails significantly in the Negroid (Congoid) and the Europoid (Caucasoid) population. Intermediate incidence data are due to racial intermixture.

Effects of lysine clonixinate and ketorolac tromethamine on prostanoid release from various rat organs incubated ex vivo.

The release of prostanoids from rat brain, gastric mucosa, lungs and kidneys incubated ex vivo has been investigated for up to 5 h after oral administration of 10 mg/kg lysine clonixinate or 1 mg/kg ketorolac tromethamine. Additionally, 60 min after drug administration, a time point of near-maximal inhibition of prostanoid release, the effects of 2.5, 10 and 30 mg/kg lysine clonixinate and of 0.0225, 0.15 and 1 mg/kg ketorolac tromethamine were compared. In all organs investigated both drugs inhibited fatty acid cyclooxygenase (COX) in a dose-dependent manner, but ketorolac tromethamine was more potent and had a longer-lasting effect than lysine clonixinate. While the ID50 values for lysine clonixinate were in the same order of magnitude for all 4 organs investigated, ketorolac tromethamine exhibited some organ selectivity with a particularly high activity in the kidneys. This effect might be related to the renal toxicity of ketorolac tromethamine. On the other hand, the difference in potency was smallest in brain suggesting that inhibition of central prostanoid biosynthesis could contribute to the rapid and effective inhibition of pain by both drugs. IC50 values for inhibition of purified COX-1 and COX-2 in vitro were slightly lower for lysine clonixinate (2.4 and 24.6 micrograms/ml, respectively) than for ketorolac tromethamine (3.7 and 25.6 micrograms/ml, respectively).

The volume protective natriuretic peptide system in the inner ear. Comparison between vestibular and cochlear compartments.

It has been suggested that the family of natriuretic peptides (NP) has a protective role in volume overloading. Specific binding sites for atrial natriuretic peptide (ANP) and the kidney analog urodilatin (URO) were identified and quantified with computerized autoradiography and biochemical assay in the cryosection in the cochlear and vestibular (utricle/ampulla) tissue. Immunohistochemistry was used to identify and localize NP-like immunoreactive cells. Different levels of specific receptors between and within the inner ear compartments were detected. The presence of specific receptors for NP, as well as unequal distribution of NP-immunoreactivity between the compartments (in certain parts of the cochlea and the endolymphatic sac), may indicate a local autocrine and/or paracrine action of these peptide systems (presumably as a result of the integration of the different peptide effects), independent of their action via the more conventional systemic route, in addition to differences in response of the inner ear compartments to the load. The present results on specific binding of ANP and URO in the inner ear tissue may suggest physiological homology between the inner ear and the kidney. Moreover, a similar role of NP in these organs is suggested.

Calcium dependent intercellular interaction of the neural crest derivate-melanocytes and the epithelial cells of the vestibular organ.

As recently established in several species, the presence of neural crest derivate-melanocytes is essential for the development of the epithelial cells regulating the ion and the potential gradients in the inner ear. The interaction between the melanocytes and the epithelial cells appears to be activated when intracellular Ca2+ increases. This may be due to a disturbance of the Ca2+ homeostasis under hydropic conditions or to the effect of the Ca-ionophore (A 23187) or hormone (alpha-MSH). Lowering of extracellular Ca2+ blocked the effect of the drug at the basal level. The interaction of the melanocytes with the epithelial or endothelial cells differs significantly. It is indicated that melanocytes may be under hormonal control and that their activation and intercellular interaction are related to the increase of intracellular Ca2+ and may be controlled by extracellular Ca2+. Furthermore, we propose that melanocytes provide a regulatory network for the maintenance of the inner ear homeostasis.

Glycerol effect on the guinea pig tectorial membrane.

The tectorial membrane (Tm) of guinea pigs has been found to have an altered organization of its matrix fibers in response to intravenously administered glycerol. Following treatment, the Tm middle zone shows an increase in waviness and clumping of fibers in non-hydropic and several hydropic ears in contrast to non-treated control ears. Residue of the internal sulcus cells occasionally fills the subtectorial space. In the present study, additional investigations were performed with scanning electron microscopy in order to study the relationship between the Tm and the organ of Corti, as well as the relationship between Hensen's stripe and the inner hair cell. Present findings provide evidence for a connection between the inner hair cell stereocilia and Hensen's stripe which may be the molecular basis for the modulation of hearing during the glycerol test in a patient with Meniere's disease.

Experimental interruption of the endolymphatic duct and its effect on the DC potential in the endolymphatic sac.

The endolymphatic sac (ES) of the guinea pig was isolated from the remainder of the inner ear by means of surgical interruption of the endolymphatic duct (ED). The DC potential in the ES lumen and the morphology of the ES were studied using glass micro-electrodes and a light microscope at various time intervals after the interruption of ED. The DC potential did not significantly change 1 h postoperatively, compared to findings in the non-operated ear, but a significant decrease of the DC potential was observed after 1, 3 and 7 days postoperatively. A histologically-stainable substance in the ES lumen was enhanced on the operated side. The isolated ES shows a disturbance of mechanism(s) maintaining the DC potential and there is a secretion of a stainable substance into its lumen.

Atrial natriuretic peptide-like immunoreactive cells in the guinea pig inner ear.

Using specific antibodies against cardiodilatin/atrial natriuretic peptide (CDD/ANP) in a conventional immuno-histochemical method (PAP) we located ANP/CDD-like immuno-reactive cells related to the secretory area, to the sensory and to the neuronal area in the compartments of the inner ear (cochlea, utricle/ampulla, and endolymphatic sac). Immunoreactive cells were unevenly distributed in the different compartments as well as within the cochlear space. Our findings suggest that ANP/CDD may play a role in the local control of fluid and electrolyte homeostasis of the inner ear. ANP/CDD-binding sites and ANP/CDD-like immunoreactivity in the inner ear may also indicate that the peptide has an additional paracrine and/or autocrine function in the organ.